Kai-Xin-San (KXS) is widely used as a traditional Chinese herbal formula for treating depression, while its regulatory mechanisms on the hypothalamic microenvironment remain unclear. This study systematically elucidated the molecular mechanisms by which KXS alleviates chronic unpredictable mild stress (CUMS)-induced depression in rats through single-cell RNA sequencing (scRNA-seq) combined with behavioral and molecular biology experiments. Behavioral tests revealed that KXS significantly ameliorated depressive-like behaviors in CUMS-induced rats. The scRNA-seq analysis of 45,482 hypothalamic cells isolated from control rats, CUMS-induced rats, KXS-treated rats and fluoxetine-treated rats revealed nine major cell types. The transcriptional atlas of these cells revealed that CUMS reduced the numbers of astrocytes and adipocytes, while increasing the number of proinflammatory microglia and endothelial cells. KXS intervention restored astrocyte subpopulations (Astro_1), upregulated the protein expression of mitochondrial metabolism-related enzyme CPT1A, reduced the M1/M2 microglial polarization ratio, suppressed the protein expression of proinflammatory cytokines TNF-α and CXCL10, and elevated the protein expression of anti-inflammatory TGF-β. KXS remodeled hypothalamic intercellular communication networks, enhancing astrocyte-neuron interactions. The protein expression of neuronal nuclear antigen, microtubule-associated protein 2, synapsin, and postsynaptic density-95 in hypothalamus was significantly increased in KXS group, which reflected changes in the number of neurons and altered synaptic plasticity. Our study is the first to demonstrate that KXS can alleviate depression through the inhibition of proactivity, restoration of astrocytic mitochondrial metabolism, and reshaping of neuro-astrocyte interaction networks to enhance synaptic plasticity through scRNA-seq in hypothalamus. These results provide a theoretical foundation for the development of hypothalamic microenvironment-targeted antidepressant strategies.Copyright © 2025. Published by Elsevier Inc.